A Critical Review of the Impact of SMARCA4 Mutations on Survival Outcomes in Non-Small Cell Lung Cancer

This critical review investigates the impact of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) mutations on survival outcomes in non-small cell lung cancer (NSCLC) through an analysis of 21 peer-reviewed articles. Survival analyses across this review demonstrated consistently worse outcomes for SMARCA4-mutated vs. SMARCA4 wild-type NSCLC patients, specifically emphasizing class 1 truncating mutations as an independent factor for poor overall survival. In addition, this review explores the clinicopathologic characteristics of SMARCA4 mutations and their impact on various treatment modalities, including immune checkpoint inhibitors (ICIs) both with and without Kirsten rat sarcoma viral oncogene homolog (KRAS) co-mutations. The potential ineffectiveness of ICI treatment in NSCLC is explored through the impact of SMARCA4/KRAS co-mutations on the tumor microenvironment. Moreover, this NSCLC review consistently reported statistically worse overall survival outcomes for SMARCA4/KRAS co-mutations than SMARCA4 wild-type/KRAS-mutated cohorts, extending across ICIs, chemo-immunotherapy (CIT), and KRAS G12C inhibitors. Designing prospective clinical SMARCA4-mutated or SMARCA4/KRAS co-mutated NSCLC trials to evaluate targeted therapies and immunotherapy may lead to a better understanding of how to improve cancer patients’ outcomes and survival rates.


Introduction
Over the past twenty years, the Food and Drug Administration (FDA) has approved a minimum of 20 new molecular entities that target oncogenic driver mutations in non-small cell lung cancer (NSCLC) [1].These targeted therapies have led to some NSCLC studies reporting a median overall survival (OS) over three years in the metastatic setting [2].While more than fifty percent of non-squamous NSCLC patients may have a mutation with an FDA-approved therapy, there remains a distinct unmet medical need for those with epigenetic dysregulation that may be more challenging to target [1,3].This need is particularly vital given that the five-year survival rate for Stage IV NSCLC patients in 2020 was only 8.2% [4].
This critical review analyzes the current literature on the impact of mutations in the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) gene, focusing on the survival outcomes for patients with NSCLC.Typically, patients with SMARCA4-deficient NSCLC clinically present with adenocarcinoma, larger invasive tumor size, a smoking history, and fewer epidermal growth factor receptor (EGFR) mutations (p < 0.05) [5,6].In addition, SMARCA4-mutated NSCLCs are associated with a higher likelihood of negative or low programmed cell death ligand-1 (PD-L1) expression (p =0.03) and higher tumor mutational burden (TMB) as compared to wild-type (WT) cases (p < 0.001) [7,8].
J. Pers.Med.2024, 14, 684 2 of 16 SMARCA4 mutations occur in approximately 5-7% of all human cancers and 7-11% of NSCLC cases [6,[9][10][11].Two classes of SMARCA4 alterations are linked to epigenetic dysregulation [12].Class 1 mutations encompass truncating mutations, fusions, and homozygous deletions, typically associated with protein loss and loss of function.Class 2 mutations involve missense mutations, which are suggested to employ dominant-negative or gain-offunction effects.Some reports indicate a loss of function leading to reduced accessibility and diminished chromatin remodeling activity, mainly observed in lung cancer [11].In a comprehensive study of 4813 tumors from NSCLC patients, Schoenfeld et al. discovered that 212 patients (4% of the total population) exhibited class 1 SMARCA4 alterations (52% of the SMARCA4 variants) [8].In contrast, 195 patients (4% of the total population) had tumors with class 2 SMARCA4 alterations (48% of the SMARCA4 variants) [8].Of particular interest, SMARCA4 mutations are associated with altered MYC gene expression, which is essential in regulating cell growth and proliferation [13].
SMARCA4 is a tumor suppressor gene located on chromosome 19p13.2that encodes the Brahma-Related Gene 1 (BRG1) protein, which is one of two mutually exclusive critical DNA-dependent ATPases (Brahma is the other) that regulates gene expression by altering the chromatin structure [14].In addition, the BRG1 protein contains a bromodomain that plays a critical role in gene transcription and is essential in recognizing acetylated lysine residues on N-terminal tails of histones [14].
BRG1 is part of the mammalian-type switch/sucrose non-fermenting (mSWI/SNF) chromatin-regulatory complex (CRC) [15].The mSWI/SNF remodeling complex is essential in regulating chromatin structures and contains approximately 10-12 subunits [16].Utilizing energy obtained through ATP hydrolysis, the catalytic subunits of human mSWI/SNF change nucleosome positioning.This alteration modulates the accessibility of transcriptional machinery to DNA, ultimately impacting the activation or repression of specific genes.The mSWI/SNF CRC protein subunits of interest are AT-Rich Interaction Domain 1A (ARID1A), SWI/SNF Related, Matrix-Associated, Actin-Dependent Regulator of Chromatin, Subfamily B, Member 1 (SMARCB1), and BRG1.They are among the most studied CRCs and have higher rates of mutations in human cancers than other CRC subunits [17].
Further research is needed to unravel the intricate interplay between SMARCA4, chromatin remodeling, and tumorigenesis in non-small cell lung cancer.A comprehensive understanding of the nuanced interplay between SMARCA4 and KRAS co-occurring mutations in the context of NSCLC is paramount, as KRAS mutations commonly co-occur with various mutations and have been found to have a poor prognosis across several NSCLC studies [18].This review aims to summarize the clinical studies that have elucidated the potential implications of SMARCA4 mutations and SMARCA4/KRAS co-mutations on survival outcomes when treating metastatic NSCLC patients across various treatments (i.e., chemotherapy and immune checkpoint inhibitors).

Materials and Methods
PubMed and the Web of Science were searched using the following keywords: "SMAR CA4" and "NSCLC".Boolean operators were used to connect specific search keywords.Peer-reviewed articles published in English between 2018 and 2023 were included, and duplicate records were removed.Articles were included if they contained any of the following concerning NSCLC patients with SMARCA4 mutations vs. SMARCA4 WT: OS hazard ratio (HR), progression-free survival (PFS) HR, median OS, or median PFS.Articles were excluded if they were solely pre-clinical or diagnostic.Articles were also excluded if the majority of SMARCA4 lung cancer patients were diagnosed with non-NSCLC (i.e., small-cell), an undifferentiated tumor, or sarcomatoid SMARCA4 NSCLC.Articles were loaded into Raayan [19] and reviewed by all authors.Discrepancies were discussed until a consensus was reached on included vs. excluded articles.
hazard ratio (HR), progression-free survival (PFS) HR, median OS, or median PFS.Articles were excluded if they were solely pre-clinical or diagnostic.Articles were also excluded if the majority of SMARCA4 lung cancer patients were diagnosed with non-NSCLC (i.e., small-cell), an undifferentiated tumor, or sarcomatoid SMARCA4 NSCLC.Articles were loaded into Raayan [19] and reviewed by all authors.Discrepancies were discussed until a consensus was reached on included vs. excluded articles.

Results and Description of Studies
In this critical review, 75 articles were identified by applying the search strategy.Twenty-eight articles were excluded and removed as duplicate records, and 47 articles were fully reviewed.Twenty-six articles were excluded due to the lack of SMARCA4-mutation NSCLC survival outcomes, primary prognostic modeling, lack of incorrect patient population (i.e., sarcomatoid histology), review articles, and being written in non-English.
Twenty-one studies were included and reviewed in this critical review of SMARCA4 NSCLC.The included articles focus on survival outcomes across various patient categories, clinicopathologic characteristics, and immune checkpoint inhibitors (ICIs) cancer treatment.Moreover, a separate subheading focusing on SMARCA4 and KRAS co-mutation survival outcomes in patients with NSCLC is included, as this has recently become a

Results and Description of Studies
In this critical review, 75 articles were identified by applying the search strategy.Twenty-eight articles were excluded and removed as duplicate records, and 47 articles were fully reviewed.Twenty-six articles were excluded due to the lack of SMARCA4mutation NSCLC survival outcomes, primary prognostic modeling, lack of incorrect patient population (i.e., sarcomatoid histology), review articles, and being written in non-English.
Twenty-one studies were included and reviewed in this critical review of SMARCA4 NSCLC.The included articles focus on survival outcomes across various patient categories, clinicopathologic characteristics, and immune checkpoint inhibitors (ICIs) cancer treatment.Moreover, a separate subheading focusing on SMARCA4 and KRAS co-mutation survival outcomes in patients with NSCLC is included, as this has recently become a medically relevant topic in the lung cancer community.Table 1 summarizes the NSCLC mutation analyzed, treatment, outcome, and relevance of each study in this review.

SMARCA4 Mutations
Schoenfeld et al. examined the clinicopathologic characteristics in a multivariate NSCLC analysis and determined that both SMARCA4-mutated class 1 (N = 149) and class 2 (N = 143) had worse OS vs. WT (p < 0.001) using Kaplan-Meir methods [8].In addition, class 1 SMARCA4-mutated patients had the worst OS compared to class 2 mutated or WT patients (N = 996) via a log-rank test analysis [8].Furthermore, an ICI survival treatment analysis (N = 87) showed an OS improvement for SMARCA4-mutated patients receiving ICI treatment vs. without ICI treatment regardless of class 1 or class 2 mutation status (p = 0.01).However, there was no significant difference in PFS (p = 0.74) or OS (p = 0.35) for SMARCA4-mutated patients receiving ICIs when comparing class 1 vs. class 2 alterations in an unadjusted analysis [8].
In a different study with the largest number of SMARCA4 mutations in this review (N = 3305), the authors focused on clinicopathologic characteristics of patients with SMARCA4 mutations and BRG1-deficient NSCLC [6].Even though this study had a large number of SMARCA4 patients, they only performed a SMARCA4 mutation PFS analysis (N = 16) with 11 patients on chemotherapy and five on CIT.These patients had extremely short PFS of only 38 and 35 days, respectively [6].
Several studies included OS multivariate analysis across stages to address the outcomes of patients with SMARCA4 mutations in the context of Stage III vs. Stage IV disease.One study conducted by Liang et al. revealed that individuals with SMARCA4 mutations experienced significantly poorer survival in both locally advanced (Stage III) (N = 21) and metastatic (Stage IV) (N = 69) scenarios when compared to those with WT status [5].The median survival for SMARCA4-mutated vs. WT patients was notably lower in Stage III (23.73 vs. 29.43months, p < 0.01) and Stage IV (11.93 vs. 28.23 months, p < 0.01) [5].To support this finding in Stage III patients, Talvitie et al.'s multivariate analysis on SMARCA4-mutated patients (N = 31) also revealed worse OS vs. WT patients (HR:1.93;p = 0.04) [23].Regardless of stage, both authors conducted a multivariate analysis showing that SMARCA4 WT (N = 221) patients had a better OS than mutated patients [(N = 105), 28.23 months vs. 12.17 months; p < 0.001] and SMARCA4-mutated patients had a worse OS than WT (HR: 3.522; p < 0.002) [5,23].
When addressing SMARCA4 mutation OS differences in a multivariate analysis of biological sex, two OS results were mixed between the sexes [5,26].Pan et al. conducted a retrospective multivariate analysis, which included a cohort across 21 medical centers.There were 44 NSCLC patients with SMARCA4 mutations, and men (N = 25) had a significantly worse OS than women (N = 19) (2.75 months vs. inestimable, HR:14.2;p = 0.02) [26].The authors shared that one reason for the inestimable survival may be the low sample size of SMARCA4 NSCLC patients.In the second study, the authors conducted a multivariate analysis, which resulted in statistically worse OS for women (N = 88) vs. men (N = 25) (HR: 4.1; p = 0.04) [5].
There were two NSCLC analyses of the OAK study involving atezolizumab.In the first OAK study analysis, Wang et al. discovered that SMARCA4 was more likely to be seen in the negative PDL-1 expression group, but there was no statistical difference in OS when comparing atezolizumab vs. docetaxel in SMARCA4-mutated (N = 100) whether the patients were PDL-1 high or negative (HR = 0.67; p = 0.256) [27].In addition, there was no difference in OS in SMARCA4-mutated NSCLC patients (N = 39) vs. SMARCA4 WT (N = 491) whether the patients were PDL-1 high or negative when treated with atezolizumab (HR = 1.185, p = 0.513) [27].In a second OAK uni-variate analysis, SMARCA4-mutated patients (N = 18) receiving atezolizumab did not fare worse than SMARCA4 WT patients (N = 181) (HR: 1.70; p = 0.064) [28].

Discussion
This SMARCA4 NSCLC critical review focuses on survival outcomes across clinicopathological characteristics, KRAS co-mutations, and treatment with ICIs within twenty-one peer-reviewed articles.Eleven of thirteen (85%) SMARCA4 mutation survival analyses in this review demonstrated significantly worse overall survival for SMARCA4-mutated NSCLC patients regardless of cancer treatment type.There were somewhat mixed results across SMARCA4-mutated treatment analysis for patients receiving ICIs and ICIs in combination with chemotherapy or CTLA-4 treatment.Three of the seven ICI analyses had worse OS for SMARCA4-mutated patients, three had no OS differences, and one outlier study highlighted that the patients with SMARCA4 mutations did better on ICIs regardless of the class of SMARCA4 mutation.Regarding SMARCA4/KRAS co-mutations, all seven treatment analyses (four with ICIs, two with non-ICIs, and one with KRAS G12C inhibitors) indicated that co-mutated SMARCA4/KRAS NSCLC patients consistently had significantly worse OS than SMARCA4 WT/KRAS-mutated cohorts [8,21,22,[34][35][36][37].

SMARCA4 Molecular Sub-Types and Genomic Features
In the largest SMARCA4-mutated survival analysis in this review (N = 407), the authors determined that class 1 truncating mutations were the strongest independent factor for significantly worse OS for NSCLC patients [8].Class 1 mutations are typically associated with BRG1 protein loss compared to class 2 mutations, not losing any BRG1 protein expression (81% vs. 0%, p < 0.001) [8].This lack of protein expression was also seen in an analysis completed at Massachusetts General Hospital (MGH), where 84% of patients with class 1 truncating mutations lacked BRG1 protein expression [6].However, class 2 mutations (missense, nontruncating) also had worse OS, indicating that protein function could be impacted even though protein expression was evident [8].
As the only outlier in this review, SMARCA4-mutated NSCLC patients treated with ICI therapy had a longer OS (p = 0.01) [8].In contrast, this ICI treatment OS benefit is not seen as appreciable when looking at SMARCA4 WT vs. class 1 or 2 SMARCA4 mutations.Alessi et al. point out that Schoenfeld's results may differ from their extensive SMARCA4 ICI therapy analysis due to imbalances in unreported baseline clinicopathological characteristics in the Schoenfeld publication, which could have altered the OS [21].
As further support, both Alessi et al. studies highlight SMARCA4 mutations as an independent factor in a significantly worse outcome for NSCLC patients [21,22].However, different conclusions arose in their two separate publication analyses of ICI treatment: (1) based on whether it was used in the first line with chemotherapy (CIT), or (2) in pretreated patients as a single agent (ICI) or ICI/CTLA4 combination therapy.In the first-line setting, SMARCA4 mutations were associated with significantly worse OS to first-line CIT treatment in non-squamous NSCLC (SMARCA4-mutated 8.1 months vs. SMARCA4 WT 15.0 months (p < 0.001) [22].In the pre-treatment setting, the authors did not find an association between SMARCA4 mutational status and ICI treatment on PFS or OS, or whether the patient had a class 1 or 2 SMARCA4 mutation [21].
When looking more granularly at truncated vs. nontruncated SMARCA4 mutations or homozygous vs. heterozygous, there was a significantly worse OS when comparing SMARCA4 homozygous truncated vs. WT (7.9 vs. 16.3 months, HR: 1.85; p < 0.0001) but no significant differences when comparing other SMARCA4-mutated categories vs. WT [11].This significant OS difference was also demonstrated when looking specifically at ICI treatment when comparing homozygous truncated vs. WT (9.9 vs. 19.5 months, HR: 1.62; p = 0.01), but no significant differences were obtained when comparing WT with the other SMARCA4-mutated categories in the ICI-treated patients [11].
TMB is a genomic feature being analyzed in NSCLC across various ICI studies [38] [23].Regardless of which stage, multivariate analyses consistently showed worse OS for SMARCA4-mutated patients, highlighting the impact of these mutations on survival outcomes.This SMARCA4 review illustrated statistically significant OS differences based on brain metastasis and mixed results based on biological sex [5,25,26].In the context of brain metastases, Miao et al. investigated clinicogenomic outcomes in ALK-positive NSCLC patients treated with alectinib, finding that SMARCA4-mutated patients with brain metastases were the only group with co-mutations to exhibit statistically worse outcomes in a multivariate analysis (HR: 8.76; p = 0.009) [25].In two multivariate analyses of the impact of biological sex on SMARCA4 mutation OS differences, one analysis demonstrated significantly worse survival for men, and the other demonstrated worse survival for women [5,26].Pan et al. reported that male NSCLC patients with SMARCA4 mutations had a markedly worse OS than females (HR: 12.64; p = 0.002), while Liang et al.'s study highlighted a worse OS for women across a cohort of Stage IV SMARCA4-mutated patients (HR:14.2;p = 0.02) [5,26].Although women had a statistically significant worse OS in the multivariate analysis, the authors share that one of the reasons for the possible large female survival discrepancy was due to lower numbers of females than males in the study (88 vs. 133) [5].
In the context of SMARCA4 NSCLC patients treated with immune checkpoint inhibitors (ICIs), one study found that those with SMARCA4 mutations had worse outcomes than WT patients regardless of ICI treatment or not (HR = 3.2; p = 0.006), and two separate OAK trial NSCLC analyses showed no OS differences with atezolizumab (ICI) vs. docetaxel in pre-treated NSCLC SMARCA4-mutated patients [27][28][29].Three additional studies demonstrated significantly worse OS outcomes for NSCLC patients with SMARCA4 mutations vs. WT patients across a multivariate analysis and various standards of care treatments [30][31][32].

SMARCA4/KRAS Co-Mutations
All seven SMARCA4/KRAS co-mutation NSCLC publications in this review showed either a significantly worse PFS or OS as compared to SMARCA4 WT/KRAS mutation analyses across standard-of-care cancer treatments or within a specific treatment analysis (i.e., KRAS G12C inhibitor, ICIs, or non-ICIs) [8,21,22,[34][35][36][37]. Two of these studies presented granular survival outcomes specific to two KRAS isoforms (G12C and G12D) [34,35].Finally, one analysis demonstrated significantly worse survival for SMARCA4/KRAS-co-mutated patients as they presented at a higher rate of Stage IV NSCLC with a significantly higher propensity of brain or liver metastasis [36].These higher rates of metastatic spread led to significantly worse survival for co-mutated SMARCA4/KRAS vs. SMARCA4 WT/KRASmutated patients who either had brain or liver metastasis (i.e., brain metastasis 7.4 months vs. 15.0 months, HR: 2.1; p = 0.0003) [36].
SMARCA4/KRAS co-mutations are among the most common co-mutations within this review.Among the largest co-mutation analyses within the review, SMARCA4/KRAS was co-mutated at a rate of 36% in NSCLC [8].Within this multi-variate analysis and across various treatments, the SMARCA4/KRAS co-mutation analysis by Schoenfeld et al.
for SMARCA4-mutated and co-mutated SMARCA4/KRAS patients to understand better how these mutations and co-mutations impact the tumor microenvironment.Utilization of this prospective biomarker approach and immune cell data analysis could inform the design of future NSCLC clinical trials.

Limitations
The vast majority of mutation and treatment analyses (i.e., single agent and combination approaches) in this review are retrospective and vary in SMARCA4 mutation sample size.The scope of the publications was limited to those in the English language published from 2018 through 2023.KRAS was the primary co-mutated gene included, rather than analyzing other possible co-mutations with SMARCA4 (e.g., KEAP1/STK11).Given these limitations, this review's strength is that it is one of the largest SMARCA4 overall survival NSCLC reviews completed to date.

Conclusions
In one of the largest reviews to date of SMARCA4-mutated overall survival data in NSCLC, two consistent trends emerged from the twenty-one publications in scope: First, SMARCA4 NSCLC mutated patients had statistically worse OS than SMARCA4 WT patients.Secondly, NSCLC patients with SMARCA4/KRAS co-mutations tended to have significantly worse overall survival than SMARCA4 WT/KRAS-mutated patients, regardless of treatment type, within each treatment analysis.Moreover, designing prospective clinical SMARCA4mutated or SMARCA4/KRAS-co-mutated NSCLC trials to evaluate targeted therapies and immunotherapy may lead to a better understanding of how to improve cancer patients' outcomes and survival rates.These findings may have implications for future research and clinical practice.

Figure 1 .
Figure 1.PRISMA flow diagram illustrating the study selection process.

Figure 1 .
Figure 1.PRISMA flow diagram illustrating the study selection process.
. It is being reviewed as a possible biomarker for the use of ICIs in SMARCA4-mutated NSCLC [21].Xu et al. showed a worse OS for NSCLC patients that are SMARCA4-mutated (TMB Low or TMB High) as compared to SMARCA4 WT/TMB High (p = 0.00019) [24].Multiple studies, including Liang et al. and Talvitie et al., conducted OS multivariate analyses across different stages, revealing consistently poorer survival for patients with SMARCA4 mutations [5,23].Liang et al.'s study demonstrated significantly worse OS in both locally advanced (Stage III) and metastatic (Stage IV) SMARCA4-mutated scenarios compared to WT status (Stage III (23.73 vs. 29.43months, p < 0.01) and Stage IV (12.17 vs. 28.23 months, p < 0.01) [5].Talvitie et al.'s analysis in Stage III patients further supported worse OS for SMARCA4-mutated patients